The use of methods for the fully automatic recording of multimodal microscopical data records essentially contributes to the successful and efficient implementation of digital technologies in microscopy. It is only by using such automated microscopes that a great number of microscopical specimens can be digitized rapidly and efficiently. In digitization, specimens—e.g., tissue sections on specimen slides—are scanned across largish areas measuring 20×20 mm2, for example. To ensure scanning with a high optical resolution—an area of 0.25 μm diameter in the specimen ought to correspond to one pixel on an array detector—, the object, or the area to be scanned, is optically divided into several smaller areas called tiles. These tiles are recorded one by one and assembled into what is called a tiled image. As a rule, however, the optimum focus position, i.e. the focus position in which the selected specimen area is focused best and imaged with the highest contrast, varies due to the specimen's topography, so that, with the focus position not being adapted, different areas are imaged with different sharpness, which mars the overall image.
The prior art discloses various methods for finding the best focus position during the automatic focusing of microscopes.
A long-established method of automatic focusing, i.e. the automatic setting of the best focus position, consists in varying the distance between the objective and the specimen surface, i.e. in shifting either the stage with the specimen or the objective along the optical axis of the objective or its elongation, which hereinafter is also called Z direction or Z axis. As a rule, distance variation is carried out in equidistant steps, with the image contrast being determined in each position. The position zF′ of the specimen stage or objective corresponding to the distance at which the image contrast in the recorded image is greatest is then chosen as focus position. To further improve focusing, one can perform interpolation between the two distances having the highest image contrast, taking additional points into account, a method known as software autofocus. This method is very simple, but has the drawback of being dependent on object textures. This means that it does not work with textureless boundary surfaces, such as, e.g., a clean glass surface or a smoothly polished metal surface. In addition, this method is relatively slow, since a great number of distances or Z positions have to be approached and images to be recorded and analyzed in each position. This makes the method unfit for, e.g., live cells.
To automatically focus on featureless surfaces, one has to make use of an autofocusing method with active illumination, a procedure known as hardware autofocus, in which, in prior art, the autofocus sensor is implemented in various ways.
A first method is that of triangulation: A light beam—as a rule, a laser beam—is directed onto the specimen surface at an angle other than normal. At least part of the light beam is reflected by the specimen surface; the site of reflection varies as the specimen is shifted along the optical axis of the microscope objective in Z direction, which can be registered by a spatially resolving light detector. However, this method cannot be used with light-scattering specimens, as a rule. Moreover, the site at which the light beam of the laser is coupled into the microscope's beam path must be specifically matched with every objective, since microscope objectives feature greatly varying pupil diameters.
Another method makes use of a confocal sensor, which is described, e.g., in US 2008/002252 A 1: A light beam—again a laser beam, as a rule—is collimated and coupled into the objective pupil collinearly with the objective axis, so that it forms a point in the focal plane. On the image side, again a point will result if the specimen—an at least partially reflecting boundary surface provided—is located in the focal plane. The farther the specimen is moved away from the focal plane, the more the image-side point will expand. The intensity loss involved in the expansion can be used as an autofocus signal. To obtain directional information as well, one can, e.g., shade half of the laser beam cross-section in the collimated segment. Alternatively, one can couple the laser beam in an off-axis mode, i.e. so that it does not coincide with the optical axis. However, this method is very sensitive to reflexivity imperfections in the specimen's boundary surface reflecting the beam, such as dirt, scratches or fissures. Where high accuracy is of the essence, the capturing area is relatively small.
The same advantages also are inherent in another method also based on the use of a confocal sensor, in this case an astigmatic sensor. Here, a cylindrical lens is additionally arranged in the beam path, and the directional information can be obtained via the astigmatic aberration.
EP 1 393 116 B1 describes a method based on the use of a tilted confocal slit: A narrow light slit is imaged onto the specimen and detected on the image side with a linear sensor that is inclined relative to the optical axis by less than 90°. Where the light slit is imaged on the linear sensor in sharp focus, the intensity is highest. The directional information can be obtained from the intensity variation on the linear sensor. Alternatively, one can tilt the slit instead of the linear sensor. This method, too, makes use of the reflection of the light beam off a boundary surface of the specimen, and therefore is sensitive to imperfections in reflexivity such as dirt, scratches and fissures. Moreover, the adjustment of slit and linear sensor, which is critical for a correct measurement, is rather laborious.
WO 2007/144197 A1 describes an autofocus system based on a tilted camera with an array sensor: Instead of a light slit, a fine grid is imaged onto the specimen and, on the image side, detected by an array sensor such as used in digital cameras, which is inclined relative to the optical axis by less than 90°. Alternatively, the grid can be inclined, and it may also be implemented as a slit pattern. What is exploited here is not primarily the intensity distribution on the sensor but rather the contrast of the grid image. This method is less sensitive to imperfections such as dirt or scratches, but greatly scattering specimens are a problem here, too, as they cannot be correctly focused on.
Another method, which is relatively fast but needs a highly complex arrangement, is based on optical coherence tomography (OCT) and a sensor designed therefore. It is an interferometric method, in which an amplitude-modulated scan (also known as A scan) is made by traversing along the Z direction at high speed and analyzing where the short-coherence radiation source creates interferences. Alternatively, the so-called Fourier domain method can be used, which works without moving parts. In this way, both light-reflecting and light-scattering specimens can be detected. As a disadvantage, this method is very sensitive to length variations in the reference beam path as well as to changes of dispersion, e.g. when objectives are changed.